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6 The Open Microbiology Journal, 2014, 8, 6-14
1874-2858/14 2014 Bentham Open
Open Access
Essential Oils, A New Horizon in Combating Bacterial Antibiotic
Resistance
Polly Soo Xi Yap1, Beow Chin Yiap2, Hu Cai Ping3 and Swee Hua Erin Lim2,*
1School of Postgraduate Studies and Research, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit
Jalil, 57000 Kuala Lumpur, Malaysia; 2School of Pharmacy, Department of Life Sciences, International Medical Uni-
versity, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia; 3School of Health Sciences, De-
partment of Chinese Medicine, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000
Kuala Lumpur, Malaysia
Abstract: For many years, the battle between humans and the multitudes of infection and disease causing pathogens con-
tinues. Emerging at th e battlefield as some of the most significant challenges to human health are bacterial resistance and
its rapid rise. These have become a major concern in global public health invigorating the need for new antimicrobial
compounds. A rational approach to deal with antibiotic resistance problems requires detailed knowledge of the different
biological and non-biological factors that affect the rate and extent of resistance development. Combination therapy com-
bining conventional antibiotics and essential oils is currently blooming and represents a potential area for future investiga-
tions. This new generation of phytopharmaceuticals may shed light on the development of new pharmacological regimes
in combating antibiotic resistance. This review consolidated and described the observed synergistic outcome between es-
sential oils and antibiotics, and highlighted the possibilities of essential oils as the potential resistance modifying agent.
Keywords: Antibiotic resistance, combination therapy, essential oils, resistance modifying agents.
INTRODUCTION
Antibiotic therapy is one of the most important therapies
used for fighting infectious diseases and has tremendously
enhanced the health aspects of human life since its introduc-
tion. Despite the advancements in this therapy, we still live
in an era where incidents of antibiotic resistant infections are
alarmingly on rise [1]. The significance of the role of antibi-
otics in nature remains unfounded due to the responses of
bacteria through the manifestation of various forms of resis-
tance following the introduction of a new antibiotic for clini-
cal use. The most important factor influencing the emergence
and spread of antibiotic resistance is the excessive bacterial
exposure to antibiotics [2]. Indiscriminate and over use of
antibiotics causes selective pressure, allowing only the fittest
genotype to thrive. Despite of the fact that evolution is inevi-
table, the intensive use of antimicrobial agents in the com-
munity, hospital and agriculture is undeniably responsible
for fuelling this crisis. Today, bacteria which are resistant
not only to a single drug but simultaneously to many drugs
are rampantly spread in the community and clinically due to
the improper use of antibiotics in the past decade [2, 3]. An-
tibiotic resistance may result in treatment failure, increased
treatment costs as well as the rate of fatalities, and creates
even broader infection control problems – spreading resistant
bacteria from hospital to community. The persistence of an-
tibiotic resistance urges the need of finding new therapies
against the multi-drug resistant bacteria.
*Address correspondence to this author at the School of Pharmacy, Depart-
ment of Life Sciences, International Medical University, No. 126, Jalan Jalil
Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia;
Tel: +60327317578; Fax: +60386567229; Email: erin_lim@imu.edu.my
Since the 1990s, rapid development in molecular biology
and high throughput screening has shed light on a more effi-
cient approach to antibiotic discovery. However, the discour-
aging outcome is that, at present, no antibiotic found by this
strategy has yet to enter clinical settings. This is b ecause too
much emphasis has been placed on identifying targets and
molecules that interact, while too little placed on the actual
ability of these molecules to permeate the bacterial cell wall,
evade efflux and avoid mutational resistance [4]. Hence dis-
covering a compound that binds to a conserved target does
not necessarily equate to finding one with antibiotic activity.
Furthermore, antibiotics with a single target are especially
vulnerable to mutational resistance.
ATTEMPTS IN NA TURAL PRODUCTS
Previously, natural products screening was almost aban-
doned, partly because it had ceased to identify new leads,
was time consuming and fitted poorly with the changing
logistics of high-throughput screening [4]. However, there is
resurgence in the use of herbal medicines worldwide. Ex-
ploitation of natural products for medicinal uses is a bloom-
ing trend nowadays. Natural products are viewed as a privi-
leged group of structures which has evolved to interact with
a wide variety of protein targets for specific purposes. Many
attempts have been made to investigate the potential role of
plant extracts and some active compound for their efficacy to
combat the problems of antibiotic resistance in bacteria.
Plant extracts consisting complex mixtures of major com-
pounds and their secondary metabolites alongside conven-
tional antibiotics exude possible synergistic effects. The ra-
tionale behind the preference for pharmaceutical combina-
Essential Oils, A New Horizon i n Combating Bacteria l Antibiotic Resistance The Open Microbiology Journal, 2014, Volume 8 7
tion is based on a long awareness that many diseases have a
complex pathophysiology. Additionally, there are many ad-
vantages of using natural products as the antimicrobial com-
pounds such as fewer adverse effects, better patient toler-
ance, and relatively inexpensive, wide acceptance due to
their traditional applications, renewability and better biode-
gradability.
Many reports have described the emergence of microor-
ganisms that are resistant to different antimicrobial agents,
the frequencies and the molecular mechanism of antibiotic
resistance involved [2, 3, 5-9]. Extracts from different parts
of plants have been widely explored in many studies for their
capability in modulating bacterial drug resistance and these
studies could serve as reference to provide possible direction
for future studies in the reversal of microbial resistance
(Table 1). Among all the articles reviewed below, almost
half of all the publications did not elucidate the mechanisms
of resistance modifying activities, indicating that this miss-
ing link should be investigated as it is imperative before
natural products can advance into clinical applications. Addi-
tionally, very few papers have given insight into the preva-
lence of the resistance reversibility by natural products, in
particular with regards to essential oils. Therefore, the pur-
pose of this mini review is to consolidate all available litera-
ture to synthesize fresh insights and to suggest potential fu-
ture research direction embarking in this area.
Plant Essential Oils
Plants produce a large array of secondary metabolites as
natural protection against microbial and pests attack, as col-
oring, scent or pollinators attractants. Essential oils, also
known as volatile oils, are products of the secondary metabo-
lism of aromatic plants. They are termed “essential” because
they represent the very essence and the most important part
of the plant. There are distinctive differences between essen-
tial oils and crude plant extracts in terms of purity, composi-
tion and the process of acquisition. Generally, essential oils
are produced through steam distillation or mechanical ex-
pression while simple plant extracts often involve the use of
solvent such as acetone, ethanol or hexane for extraction.
During distillation, water condensate is separated by gravity
leaving a very small amount of volatile liquid that is the es-
sential oil. Hence, they are extremely concentrated due to the
nature of the extraction process. According to the National
Cancer Institute, oils produced by means of chemical sol-
vents are not considered true essential oils as the solvent
residues can lead to alteration of the purity and fragrance of
the oils [19]. Technically, essential oils are not true oils as
they do not contain lipid content instead they are highly
complex volatile compounds which consist of about 20-60
components in various concentrations. The components
comprise of two biosynthetically related groups namely ter-
penes and aromatic compounds. In this multi-component
mixture, two or three major components are present at rela-
tively high concentrations (20-70%) compared to other com-
ponents which are present in trace amounts [20]. For in-
stance, the main component of clove (Syzygium aromaticum)
essential oil is eugenol (68.52%) while -caryophyllene
(1.85%) is present in trace amounts [21]. Other major com-
ponents present in essential oils are terpinen-4-ol (30.41%)
of marjoram (Origanum majorana L.) essential oil, thymol
(57.7%) of Thymus vulgaris essential oil, bicyclogermacrene
(26.1%) and -caryophyllene (24.4%) of Lantana camara L.
essential oil, -thujone (41.48%) of Salvia officinalis L. es-
sential oil, and -(-)-bisabolol (63%) of Eremanthus ery-
tropappus essential oil [22-26].
Table 1. List of antibiotic resistance modifying plant extracts against a panel of microorganisms.
Plant family name Part Used Microorganisms Modulatio n of
Resistance Method of Study References
Rosmarinus officina lis Aerial part S. aureus MDR efflux inhibition
Ethidium bromide
efflux assay [10]
Lycopus europaeus N/A S. aureus - - [11]
Fissistigma cavaleriei Root P. aeruginosa -lactamase inhibi-
tion
-lactamase
inhibitory assay [12]
Cardiospermum grandiflorum Leaves S. aureus - - [13]
Momordica charantia L. Leaves MRSA Efflux pump
inhibition
Efflux pump
inhibitory assay [14]
Mentha arvensis L. Leaves E. coli - - [15]
Turnera ulmifolia L. Leaves MRSA - - [16]
Catha edulis Leaves
Streptococcus oralis,
Streptococcus sanguis, Fuso-
bacterium nucleatum
- - [17]
Punica granatum Fruit MRSA Efflux pump inhibiton
Time-kill assay,
-lactamase production
detection, ethidium
bromide efflux assay
[18]
8 The Open Microbiology Journal, 2014, Volume 8 Yap et al.
Various essential oils have been reviewed to possess dif-
ferent biological properties such as anti-inflammatory, seda-
tive, digestive, antimicrobial, antiviral, antioxidant as well as
cytotoxic activities [20, 27]. These findings highlight an ex-
citing scientific interest whereby essential oils warrant spe-
cial attention because they represent a distinctive group of
possible novel drug compounds due to their chemical and
structural variance that makes them functionally versatile.
Due to their chemical diversity, the ongoing hypothesis is
whether their biological effects are reflected only in the main
molecules at the highest levels according to the composi-
tional analysis or that these biological effects arise from the
synergism of all molecules present. In most cases reviewed,
only the main constituents of certain essential oils such
eugenol, thymol and carvacrol were analyzed [28, 29]. Sev-
eral reports have demonstrated that these compounds exhib-
ited significant antimicrobial activities when tested individu-
ally [26, 30]. Dorman and Deans (2000) demonstrated that
the individual oil components (mainly with phenolic struc-
tures) were able to exhibit a wide spectrum of antibacterial
activity and that the chemical structures greatly affect the
components effectiveness and their mode of antibacterial
action [31]. Bassole et al. (2010) pointed out the synergistic
effects on the growth inhibition of Listeria monocytogenes,
Enterobacter aerogenes, Escherichia coli and Pseudomonas
aeruginosa in eugenol/linalool and eugenol/menthol combi-
nations [32]. Although the biological properties of essential
oils are found to be closely related with the major compo-
nents of the oils, the amplitude of their effects could be at-
tributed to their high concentration comprised in the original
oil, masking the effects of minor components or when the
high concentration components were isolated and tested
alone. Thus, interactive functions of the various components
contained in an essential oil, in comparison to the action of
one or two main components of the oil seem unresolved. The
other side of the coin is that whole essential oils exert greater
antibacterial activity compared to the major components
alone [27]. It has also been postulated that the function of the
main components is regulated by other minor molecules
which help in potentiating synergistic effect [32]. It is likely
that several components in essential oils play a role in char-
acterizing the fragrance, the density, the texture, the color,
ability in cell penetration, lipophilicity, fixation on cell
walls, and most importantly the bioavailability. Considering
that a vast range of different groups of chemical compounds
are present in one essential oil, it is most lik ely that antibac-
terial activities cannot be attributed to one specific mecha-
nism or component; and hence, there may be several targets
in a cell which result in the potentiating influence. Thus, it is
more meaningful and rational to study the whole essential oil
rather than some of its components as whether concept of
synergism truly exists between the components in essential
oils [33].
COMBINATION OF ANTIBIOTICS AND ESSENTIAL
OILS TO REVERSE RESISTANCE
In combination therapy, synergy is said to occur when
the combined effect is greater than the sum of the individual
effects. An additive effect is observed with the combined
effect which is equal to the sum of the individual effects.
Indifference is observed when there is no interaction be-
tween one another. Antagonism is defined when the com-
bined effect is less than when the two compounds are indi-
vidually applied. The reversal of resistance is said to occur
when a synergistic outcome is observed. As an example, one
of the strategies employed was to screen against -lactamase
producers and by mechanism-based inhibition of the active-
site serine hydrolases for compounds that can antagonize the
antibiotic-destroying hydrolases. Clavulanic acid (sulbactam
or tazobactam) from a streptomycete in combination with
amoxicillin was the outcome of this approach [34]. However,
the victory against bacterial resistance did not last long; the
frequent use of clavulanic acid has led to the emergence of
resistant variants lik e any other of its antib iotic ancestors
[35]. As resistant b acterial strains will eventually emerge in
response to widespread use of a particular antibiotic and
limit its lifetime, knowledge of the principal and specific
resistance mechanisms provides scientists the insights into
strategies for development of new therapeutics. In the p ast,
when resistance to a -lactam antibiotic occurred, pharma-
ceutical scientists modified the periphery of the -lactam
warhead to obtain a more effective variant and that was how
the second- and third generation -lactams of both th e peni-
cillin and cephalosporin emerged.
Combination between conventional antimicrobial agents
and essential oils is a new concept; a few examples are de-
scribed (Table 2). Sometimes, essential oils have been found
to be synergistic enhancers in that though they may not pro-
duce any significant inhibitory effects when used alone, but
when they are used in combination with the standard drugs,
the combinatory effect surpasses their individual perform-
ance and produces enhanced antimicrobial activity [11].
Synergistic activity exerted using essential oils has been
found to reduce the minimum effective dose of antibiotics in
the treatment of infections. This reduces the adverse effects
of the antibiotic. Most importantly, association of antibiotics
with essential oils targeting resistant bacteria may have dif-
ferent mechanism of action and it may lead to new choices to
overcome the onslaught of microbial resistance. Exploitation
of essential oils in preventing bacterial resistance is believed
to be more promising because essential oils are multi-
component in nature compared to many conventional antim-
icrobials that only have a single target site.
Pelargonium graveolens essential oil was reported to re-
duce the minimum effective dose of norfloxacin against Ba-
cillus cereus, Bacillus subtilis, Escherichia coli, and Staphy-
lococcus aureus [30]. Antibiotic modifying capacity of Lan-
tana camara L. essential oil on amikacin against S. aureus
and P. aeruginosa by gaseous contact was demonstrated by
Sousa et al. (2012). The microorganisms were exposed to the
volatile constituents by indirect contact during the disk diffu-
sion test and the amikacin activity was reported to have in-
creased by 65% [36]. Rodrigues et al. (2009) reported that
the essential oil of Croton zehntneri leaves is able to enhance
the gentamicin activity by 42.8% against P. aeruginosa
through gaseous contact suggesting that the oil possesses a
potential to be used as an adjuvant in antibiotic therapy [37].
These promising investigations indicate that the combination
of essential oils with conventional antibiotics provides prom-
ising and significant potential for the development of novel
therapeutics and treatment of infectious diseases caused by
multidrug-resistant microorganisms. There is a need fo r
Essential Oils, A New Horizon i n Combating Bacteria l Antibiotic Resistance The Open Microbiology Journal, 2014, Volume 8 9
Table 2. List of essential oils/antibiotics combinations showing combinatory effects against a panel of microorganisms.
Pair combinations Microorganisms Methods Interaction References
Eremanthus erythropappus/ ampicillin S. aureus Time-kill assay Synergistic [37]
Oregano/ fluoroquinolones
Oregano/ doxycycline
Oregano/ lincomycin
Oregano/ maquindox
E. coli Broth microdilution
Checkerboard assay Synergistic [38]
Pelargonium graveolens/ norfloxacin S. aureus, B. cereus Agar dilution Checkerboard assay Synergistic [30]
Lantana montevidensis/ aminoglycosides E. coli Broth microdilution
Checkerboard assay Synergistic [36]
Eugenol/ vancomycin
Eugenol/ -lactams
E. coli, E. aerogenes, P. vulgaris, P.
aeruginosa, S. typhimurium
Broth microdilution Checkerboard
assay Synergistic [28]
Croton zehntneri/ gentamicin S. aureus, P.aeruginosa Disk diffusion test
(indirect contact of EO) - [37]
Rosmarinus officinalis/ ciprofloxacin K. pneumoniae Broth microdilution
Checkerboard assay Synergistic [39]
Eucalyptus/ chlorhexidine digluconate Staphylococcus epidermidis Broth microdilution
Checkerboard assay Synergistic [40]
Zataria multiflora/ vancomycin S. aureus (MRSA and MSSA) Broth microdilution
Checkerboard assay Synergistic [41]
Aniba rosaeodora/ gentamicin
Pelargonium graveolens/ gentamicin
Bacillus cereus, Bacillus subtilis,
S. aureus, E. coli, Acinetobacter
baumannii, Serratia marcescens,
Yersinia enterocilitica
Broth microdilution
Checkerboard assay Synergistic [30]
Citrus limon/ amikacin
Cinnamomum zeylanicum/ amikacin Acinetobacter spp Broth microdilution
Checkerboard assay Synergistic [42]
Coriander/ chloramphenicol
Coriander/ ciprofloxacin
Coriander/ gentamicin
Coriander/ tetracycline
A. baumannii Broth microdilution
Checkerboard assay Synergistic [43]
MRSA, methicillin-resistant S. aureus; MSSA, methicillin-sensitive S. aureus.
more studies concerning the molecular basis of synergistic
interactions, in order to further understand the synergistic
mechanism which is fundamental for this continuing search.
THE PREVALENCE OF RESISTANCE MODIFYING
CAPABILITY BY ESSENTIAL OILS
Antimicrobial agents are classified based on their princi-
pal mechanisms of action. These mechanisms include inter-
ference with cell wall biosynthesis (-lactams and glycopep-
tides agents), inhibition of bacterial protein synthesis (mar-
colides and tetracyclines), interference with nucleic acid syn-
thesis (fluroquinolones and rifampin), inhibition of a meta-
bolic pathway (trimethoprim-sulfamethoxazole, and disrup-
tion of bacterial membrane structure (polymyxins and dap-
tomycin) [9]. Three main targets of antibiotics are cell wall,
protein and nucleic acids biosynthesis. Throughout the years
since antibiotic was introduced, bacteria have acquired vari-
ous resistances to survive in the deluge of antibiotics. The
mechanisms vary and make the work of mitigating the
spread of resistance more challenging.
-lactam antibiotics are the commonest treatment for
bacterial infections. This antibiotic class consists of four
major groups: penicillins, cephalosporins, monobactams and
carbapenems. Hydrolysis of -lactam compounds by -
lactamases is the most widespread mechanism of bacterial
resistance against this class of antibiotic. Capability of
Staphylococcus aureus in producing penicillinase (a form of
-lactamase) to destroy penicillin G was reported within two
years after the antibiotic was first introduced [44]. The oc-
currence of MRSA is a classic example of the redundancy of
new antimicrobials with regards to only one species.
Beta-Lactamase Inhibition
The -lactamases are the major defense of gram-negative
bacteria against -lactam antibiotics. Under the selective
pressure of -lactams, bacteria produce a vast array of -
lactamases. Bacteria respond with a plethora of “new” -
lactamases – including extended-spectrum -lactamases
(ESBLs), plasmid-mediated AmpC enzymes, and carbap-
enem-hydrolyzing -lactamases (carbapenemases) with vari-
able successes [45]. Resistance is typically mediated by the
expression of plasmid-encoded -lactamases, such as TEM-
1, TEM-2, or SHV-1, which hydrolyze and inactivate these
drugs. However, investigations into essential oils/antibiotics
combination against beta-lactamase producers are currently
10 The Open Microbiology Journal, 2014, Volume 8 Yap et al.
limited, there is one report on the synergistic effects of oreg-
ano essential oil in combination with fluroquinolones, doxy-
cycline, lincomycin, maquindox and flofenicol against
ESBL-producing E. coli suggesting the possibility that es-
sential oils might function as the ESBLs inhibitor [38].
Bacterial Efflux Pump Inhibition
Most of the antibiotics need to be transported across the
cell membrane and subsequently achieve an effective con-
centration in the cytoplasm to exert inhibitory effects on bac-
teria. It was demonstrated by Walsh that the overproduction
of protein pumps at the bacterial membrane facilitates the
pumping out of the drug to be faster than it can diffuse in to
keep intra-bacterial drug concentrations below th e therapeu-
tic level [8]. The pumps are highly conserved and are chro-
mosomally encoded elements while multidrug resistance
efflux pumps are variants of membrane pumps in all micro-
organisms in response to the external environment. They are
able to efflux a large range of compounds including syn-
thetic antibiotics that were not present in the natural ecosys-
tems before their synthesis by humans. Thus, it is believed
that bacteria will not easily resist compounds which are natu-
ral as compared to the synthetic compounds (the later classes
of antibiotics). Related study on phytochemicals addresses
this hypothesis and they have been proven to be potent an-
timicrobial agents. The ability of essential oils in inhibiting
the multidrug efflux pump reveals its potential in broader
spectrum of pump-inhibitory activity against multi-drug re-
sistant organisms [10]. Lorenzi et al. (2009) evaluated that
general component in the essential oil of Helichrysum itali-
cum not only reduces chloramphenicol resistance of the
multi-drug resistant Enterobacter aerogens that overex-
presses efflux pumps but also modulates the intrinsic resis-
tance of the wild-type control strain and other gram-negative
bacteria [46].
Cell Wall and Membrane Disturbance
The bacterial cell wall biosynthetic machinery remains
one of the most promising niches for antibiotic targets. How-
ever, the impermeable nature of the gram-negative envelope
and presence of multiple efflux pumps in combination with
other resistance mechanisms contribute to the difficulty of
this task. Clinical resistance to -lactams in gram-negative
bacteria is often coupled with reduced outer membrane
permeability. As the secondary constituents of the aromatic
plants, essential oils are known to contain wide ranges of
polyphenols and terpenoids. These phenols possess a strong
binding affinity to different molecular structures such as pro-
tein or glycoproteins due to their large lipophilicity. Hence,
they have great affinities for cell membranes and exhibit
high potential to permeate through cell walls, leading to the
leakage of cell contents [28, 47]. The ability of tea tree oil
(Melaleuca alternifolia) acting as membrane permeabilizer
leading to loss of chemiosmotic control in both gram-
positive and gram-negative bacteria was elucidated by Cox
et al. (2000) [48]. Later, the biological damage of tea tree oil
on cell ultra-structures (i.e. cytoplasm and cytoplasmic
membrane) was also studied with the aid of electron micros-
copy [49, 50].
Anti-Quorum Sensing
The role of quorum sensing is well known in microbial
pathogenicity and antibiotic resistance. Quorum sensing is
responsible for motility and swarming patterns, biofilm for-
mation and stress resistance based on the signaling of mole-
cules. An example of a well-studied molecule in this area is
acylated homoserine lactones (AHLs). The Gram-negative
bacteria use AHLs for signaling whereas the Gram-positive
bacteria use modified oligopeptides. The crucial role of quo-
rum sensing on so many essential aspects of the bacterial
ecology makes this an interesting process to target to control
persistent infections due to antimicrobial resistance.
Screening of potential quorum-quenching activities often
involves biosensors and bioluminescene production or inhi-
bition. Some of the common biosensors include Chromobac-
terium violaceum CV026 and N-acyl homoserine lactone
producing E. coli [51-54]. Rose, geranium, lavender, clove
and rosemary oils were found to be the QS inhibitors in the
sensor strains Chromobacterium violaceum CV026, Es-
cherichia coli ATTC 31298, Chromobacterium violaceum
(CV12472 and CVO26) and Pseudomonas aeruginosa
(PAO1) respectively [52, 55].
Sensitivity of Gram-Positive and Gram-Negative Bacte-
ria Towards Essential Oils
Generally, essential oils are more efficacious towards
gram-positive than gram-negative bacteria [56, 57]. It has
been hypothesized that the presence of lipopolysaccharide
encompassing the bacterial peptidoglycan layer has restricted
the diffusion of hydrophobic compounds into the cytoplasm
[58]. However, not all studies of essential oils have con-
cluded that gram-positives are more susceptible [22, 59].
Interestingly, in a study carried out by van Vuuren et al.
(2009), combination of the essential oil of Rosmarinus of-
ficnalis with ciprofloxacin against gram-positive bacteria
gave an antagonistic profile while Rosmarinus of-
ficnalis/ciprofloxacin against gram-negative bacteria dis-
played a favorable synergistic profile [39]. The prevalent
antagonistic interaction test against S. aureus also suggested
that natural therapies using essential oils should be moni-
tored carefully when combined with antibiotics. Since only
inadequate sample sets have been studied, more exhaustive
investigations would warrant a better potentiating profile of
essential oils as resistance modifiers of antibiotics in clinical
applications.
STRATEGIES TO BYPASS THE OUTER MEM-
BRANE BARRIER IN MULTIDRUG RESISTANT
GRAM-N EGATIVE BACTERIA
In Gram-negative bacteria, the cell envelope comprising
an outer and an inner membrane is a sophisticated macro-
molecule assembly protecting the cell against extracellular
toxic compounds. Membrane proteins, namely the porins are
involved in regulating the internal accumulation of various
hydrophilic molecules including the antibiotics through pas-
sive diffusion. The outer membrane of Gram-negative bacte-
ria plays a crucial role in providing an extra layer of protec-
tion to the bacteria as a selective barrier. Alterations of com-
position of outer membrane and porins resulting in reduced
permeability in beta-lactam antibiotics have been demon-
strated widely in clinical resistant isolates [60]. The exis-
tence of large number of antibiotic resistant bacteria species
due to this type of mechanism highlights the importance of
the outer membrane barrier in antibiotic sensitivity.
Essential Oils, A New Horizon i n Combating Bacteria l Antibiotic Resistance The Open Microbiology Journal, 2014, Volume 8 11
Lipopolysaccharide (LPS) is the major component of the
outer membrane of Gram-negative bacteria. LPS contributes
greatly to the structural integrity of the bacteria and also in-
creases the negative charge of the cell membrane due to the
charged sugars and phosphate present in the polysaccharide
[61]. Despite the role of LPS in creating a permeability bar-
rier, the presence of porin proteins in the outer membrane
permits passage of molecules smaller than 600 daltons [61,
62].
Destabilization of LPS Barrier and Increase of Antibiotic
Influx
As discussed by Bolla et al. (2011), an alternate possibil-
ity to facilitate the antibiotics to gain access into the cells is
to bypass the outer membrane barrier [63]. To bypass the
barrier through LPS modifications, chemical facilitators such
as detergents, chaotropic agents and polymyxines have been
proposed [64, 65]. It is noted that only very limited studies
have been reported on the molecular basis of the synergy
efficacy between antibiotics and the membrane-active agents
and also the study parameters.
To circumvent the membrane barrier, destabilization of
LPS layer using detergents or chaotropic agents was pro-
posed. Treatment by Tris/EDTA results in massive release of
LPS packing and thus facilitates the diffusion of hydrophilic
compounds through the membrane lipid barrier. It has been
demonstrated that the LPS-disturbed bacteria are more sus-
ceptible to antibiotics [66]. However, the major adverse ef-
fect of using these chaotropic agents and detergents is their
high toxicity on biological membrane, consequently leading
to the medical safety issues. Hence, it is valuable to explore
the possibility of natural products being potential membrane
permeabilizer in restoring the efficacy of the existing antibi-
otics particularly beta-lactams. As demonstrated by Hurdle et
al. (2011), membrane-damaging agents may interfere with
multiple targets through the interaction of lipophilic moiety
with the bacterial membrane, through alteration of the proton
motive force, which in turn leads to leakage of cytoplasmic
content and eventually cell death [64].
Cell Targets of the Natural Membrane-Active Agents
Natural products have been found to have great effects in
disrupting the bacterial membrane [67]. It is likely due to the
presence of lipophilic compounds such as cyclic hydrocar-
bons, terpenes and aromatics which are abundantly found in
the aromatic plants [68]. Broadly, site(s) of toxic action can
be divided into (i) changes in membrane structure, and (ii)
changes in membrane function. Specifically, changes in
membrane function often involve the changes in energy
transduction and changes in activity of membrane-bound
enzymes [68].
In the work of Wilson et al. (2001), it is highlighted that
the bacterial surface physiology could seek better under-
standing using available instrumentation through the meas-
urement of zeta potential [69]. The bacteria invest a signifi-
cant portion of their metabolic energy in the synthesis and
maintenance of the components of the cell surface further
supporting the idea of interfacial physiology’s importance to
the general well being of the microorganism [70]. It is vital
to understand that the bacterial cell surface is related to the
disparate physiological functions such as envelope diffusion,
shape maintenance, cell growth and division. It is demon-
strated that Gram-negative bacteria have a more negative
surface charge than the Gram-positive ones [71]. It is noted
that a standardized methodology for evaluating the mem-
brane permeabilizing activity is lacking. Consequently, the
activity of the compounds tested in unrelated studies cannot
be compared directly. Table 3 summarizes the site of actio n
of essential oils on the membrane.
IN VITRO EVALUATION OF SYNERGY TESTING
Currently, there is no clear regulation or standardization
of the methodology to evaluate the inhibitory activity of es-
sential oils as well-established as for antibiotics [14]. Fo r
antibiotics, various methods have been employed by re-
searchers to determine the minimum inhibitory concentration
(MIC): disk diffusion test, agar dilution test and broth mi-
crodilution test. A number of methods are used to detect
synergy as well. The checkerboard and time-kill curve meth-
ods are the most widely used techniques and the former is
relatively easy to perform and monitor.
The main disadvantage of the results of in vitro studies is
that it is difficult to carry out comparison among each of the
study because of the different test methods, different meth-
ods of extraction of essential oils, test assays, and variation
in chemical phytoconstituents in plants due to different agro-
climatic conditions, harvesting seasons and plant phenotype.
All these factors will influence the phytochemicals present in
the essential oils in a considerable manner [15]. In addition,
culture conditions have a predominant influence in the stud-
ies; and therefore these should be precisely stated in reports.
In vitro susceptibility of a clinical isolate to a particular
antibiotic does not guarantee the success of the clinical use
of the therapeutic agent. The clinical outcome depends on a
wide range of other factors such as the site of infection,
pharmacological properties of the antibiotic, concomitance
of other diseases and efficiency of specific and non-specific
defense mechanism. Thus, in vitro susceptibility testing is
necessary but not sufficient for a positive clinical decision.
BACTERIAL RESISTANCE TO ESSENTIAL OILS
Interest in essential oils as potential therapeutics to eradi-
cate antibiotic resistance has been increasing and the rising
concern is whether the bacterial tolerance to the essential oil
components would be induced when these compounds are
used clinically on a large scale. The extent of bacteria in ac-
quiring resistance to essential oil components has yet to be
systematically and extensively investigated. Limited studies
have been carried out while much focus has been placed on
identifying the novel compound as resistance modifier and
expanding the phytopharmaceutical library. Though the tea
tree oil has been approved for medicinal use in Australia
since 1920s, clinical resistance to the essential oil has not yet
been reported [73]. In an in vitro resistance induction study,
resistant sub-population of MRSA was detected when the S.
aureus was repeatedly exposed to tea tree oil for several
generations [74]. Conversely, in two other studies using dif-
ferent parameters to study changes in susceptibility of the
multiple antibiotic resistance phenotype bacteria, little
12 The Open Microbiology Journal, 2014, Volume 8 Yap et al.
Table 3. Cell ta rgets of membrane-active compounds and its mode of study.
Targets Mode of S tudy Substances References
Cell morphology:
Alteration of cell shape or surface struc-
ture of the cell
Scanning electron micrograph
(SEM)
Cudrania tricuspidata EO; Allium sativum EO;
oregano EO; eugenol; epigallocatechin gallate
[72-76]
Transmission electron micrograph
(TEM) Tea tree oil; Fortunella crassifolia EO [47, 50]
Cytopla smic membrane:
Alteration of integrity and permeability K+ leakage assay Tea tree oil [48, 77]
Respiration assay Tea tree oil [48, 77]
Propidium iodide uptake assay Ferulic and gallic acids [28, 48, 78]
Cell wall OM permeability test Ceratotoxin A; luteolin; flavonoids isolated
from smaller galangal [79-81]
Cell lysis assay Oregano, thyme, clove EOs [82, 83]
Cell surface charge Zeta potential measurement Ferulic and gallic acids; lipids [78, 84]
evidence was provided to suggest the occurrence of resis-
tance to tea tree oil [85, 86]. In a single-step mutant resistant
study, mutant resistant to tea tree oil was undetectable at 2 x
MIC for the all S. aureus isolates [87]. A similar lack of sig-
nificantly increased MICs was observed on the development
of single- and multistep antibiotic resistance in S. aureus and
E. coli against tea tree oil [88]. A study involving passaging
bacteria up to fifty times with sequential exposure of oreg-
ano and cinnamon essential oils was reported. Out of the 48
clinical isolates and 12 reference strains under study, only
Serratia marcescens, Morganella morganii, and Proteus
mirabilis treated with oregano increased their resistance to
this essential oil. No resistance to cinnamon bark oil was
reported [89].
Overall, these studies provide limited evidence to suggest
the spontaneous occurrence of essential oils resistance. It is
likely that the multi-component nature of essential oils may
reduce the potential of the occurrence of essential oils resis-
tance because numerous targets need to adapt to hamper the
effects of the essential oils. In addition, if membrane perme-
abilizing effects are one of the modes of action for the essen-
tial oil, it is unlikely to that the resistance will develop spon-
taneously. As discussed by Langeveld et al. (2013), chang-
ing membrane structures and/or composition instantaneously
arrests the viability of the bacteria [67]. Hurdle et al. (2011)
also suggested a low potential for the development of resis-
tance on membrane-active agents due to the LPS modifica-
tion systems [64]. Issues of potential resistance remain if
essential oils make their way into clinical applications, par-
ticularly against the multidrug resistant microorganisms.
CONCLUSION
The reason for research in reversal of antibiotic resis-
tance, broadly, is to preserve a healthy microbial ecosystem
surrounding humans. The emergence of antibiotic resistance
clearly demonstrates the dynamic evolution of microorgan-
isms’ response to the hostile environment of antibiotics.
Mastering the evolutionary trajectories of the microbial
pathogens would aid in preventing their emergence and dis-
semination.
As discussed in the review, there are plenty of possibili-
ties for the essential oils to be used in comb ination with anti-
biotics as new treatment modalities to the bacterial infec-
tions. Despite the promising results given by pharmacologi-
cal in vitro studies so far, there are problems that still need to
be addressed such as stability, selectivity and bioavailability
of these natural products in the human body and any possible
adverse herb-drug interaction. Additionally, the optimal ratio
and dosing regimens should be explored for higher efficacy
and decreased toxicity. These critical parameters have to be
established in order to provide sufficient evidences to coast
through the phases of clinical trials. Or else, new discoveries
on bench top, however impressive, will never be translated
into drugs for clinical applications.
The exploitation of essential oils as a potential replace-
ment therapy represents ‘a new era of phytopharmaceuti-
cals’. Hopefully, the present promising results will open the
door for more research into the related field, gain momentum
and hasten the process; the discovery will be faster than the
evolution of bacteria. The best way is to make full use of the
advancement that is available in this era, in the hope that
conventional natural products discovery by means of high
throughput analysis would shed light on modern drug dis-
covery. Perhaps, in the future, essential oils can progress
from being one of the traditional curative agents to become a
widely used therapy in the modern medical domain.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no con-
flicts of interest.
ACKNOWLEDGEMENTS
Declared none.
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Received: October 21, 2013 Revised: December 24, 2013 Accepted: December 26, 2013
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